title: Characterization and application of a flow system for in vitro multispecies oral biofilm formation.
abstract: OBJECTIVE
  Bacteria in the oral cavity grow in the form of biofilms; these structures are subject to constant saliva or gingival crevicular fluid flow conditions. The aims of this study were: (i) to develop and to characterize an in-vitro biofilm model with oral bacteria growing under flow and shear conditions; and (ii) to demonstrate the usefulness of the model for evaluating the activity of three antiplaque agents.
  
  
  METHODS
  We used a bioreactor to grow the oral bacteria Streptococcus oralis, Actinomyces naeslundii, Veillonella parvula, Fusobacterium nucleatum, Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis under planktonic conditions. Biofilms were established using a modified Robbins device on hydroxyapatite (HAP) discs. Three- to 7-d-old biofilms were analysed using culture methods, scanning electron microscopy, Live/Dead staining and fluorescence in-situ hybridization (confocal laser scanning microscopy). Finally, we assessed the antimicrobial activity of three mouthrinses [0.12% chlorhexidine (CHX), 0.12% chlorhexidine and sodium fluoride (CHX+NaF) and 0.12% chlorhexidine and 0.05% cetylpyridinium chloride (CHX+CPC)] using a planktonic test (short interval-killing test) and in our 4-d biofilm model.
  
  
  RESULTS
  The viable cell counts showed that each species was consistently found in the biofilms throughout the study. The architecture and cell distribution were similar to those described for biofilms in situ, with the exception of a thin layer of living cells that was found close to the HAP. The effectiveness test of the mouthwashes demonstrated that cells in biofilms showed more tolerance compared with planktonic cells. Moreover, it was observed that in 4-d biofilm formed in vitro, CHX+CPC caused significantly higher mortality compared with CHX (p = 0.003) and CHX+NaF (p < 0.001).
  
  
  CONCLUSIONS
  Our results suggest that we have a highly reproducible system for multispecies oral biofilm formation and that it is a useful tool for assessing antibacterial molecules before their clinical evaluation. It also has great potential to be used in basic research on supragingival and subgingival biofilms.
authors.affiliation: Department of Microbiology, DENTAID S. L., Cerdanyola del Vallès, Spain.
authors.forename: V
authors.initials: V
authors.lastname: Blanc
authors.forename: S
authors.initials: S
authors.lastname: Isabal
authors.forename: M C
authors.initials: MC
authors.lastname: Sánchez
authors.forename: A
authors.initials: A
authors.lastname: Llama-Palacios
authors.forename: D
authors.initials: D
authors.lastname: Herrera
authors.forename: M
authors.initials: M
authors.lastname: Sanz
authors.forename: R
authors.initials: R
authors.lastname: León
mesh_terms: D000190:Actinomyces / Q000254:growth & development; D016976:Aggregatibacter actinomycetemcomitans / Q000254:growth & development; D000891:Anti-Infective Agents, Local / Q000494:pharmacology; D058491:Bacterial Load / Q000187:drug effects; D001431:Bacteriological Techniques; D018441:Biofilms / Q000254:growth & development*; D019149:Bioreactors; D002594:Cetylpyridinium / Q000494:pharmacology; D002710:Chlorhexidine / Q000494:pharmacology; D017886:Durapatite / Q000737:chemistry; D016967:Fusobacterium nucleatum / Q000254:growth & development; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008826:Microbial Sensitivity Tests; D050296:Microbial Viability; D018613:Microscopy, Confocal; D008855:Microscopy, Electron, Scanning; D009055:Mouth / Q000382:microbiology*; D009067:Mouthwashes / Q000494:pharmacology; D016966:Porphyromonas gingivalis / Q000254:growth & development; D012463:Saliva / Q000502:physiology; D012969:Sodium Fluoride / Q000494:pharmacology; D019533:Streptococcus oralis / Q000254:growth & development; D014678:Veillonella / Q000254:growth & development
chemical_list: D000891:Anti-Infective Agents, Local; D009067:Mouthwashes; D012969:Sodium Fluoride; D017886:Durapatite; D002594:Cetylpyridinium; D002710:Chlorhexidine
keywords: biofilm model; chlorhexidine; confocal laser scanning microscopy; oral bacteria